P426: HDAC INHIBITORS RESTORE SENSITIVITY TO VENETOCLAX AND NAVITOCLAX IN AML CELLS RESULTING IN THE IRREVERSIBLE DNA DAMAGE
Authors: Elmira Khabusheva, Mahesh Tambe, Juho Miettinen, Caroline Heckman
Journal: HemaSphere (2023)

Background: The BCL-2 inhibitor venetoclax, in combination with hypomethylating agents (HMA) or low dose cytarabine, has been introduced to clinical practice as an alternative for AML patients unfit for intensive chemotherapy. However, ~30% patients fail to achieve complete remission with venetoclax-based treatment and develop resistance. Known mechanisms of venetoclax resistance include acquired BCL-2 mutations and gene amplification, upregulation of MCL-1 and BCL-xL, activation of pro-survival MAPK signaling, epigenetic profile alteration, and TP53 mutation. Knowledge of venetoclax resistance mechanisms could be applied for the development of novel combinational strategies. Aims: Within this work we aimed to identify drugs that can enhance sensitivity to venetoclax in relapsed AML and provide insights into the mechanisms mediating the activity of novel venetoclax-based drug combinations. Methods: To identify potential drug combination partners for venetoclax and the BCL-2/BCL-xL inhibitor navitoclax, we used ex vivo drug screening data (N = 427 drugs) for 53 samples from patients with relapsed AML and 51 samples from patients with newly diagnosed AML. Drug sensitivity scores (DSS) based on the area under the curve were used to evaluate the sensitivity of the AML samples to each drug and correlate with DSS for venetoclax and navitoclax. Genome-wide CRISPR data from DepMap was used to study genetic vulnerabilities of AML cells with differential sensitivity to venetoclax. BeatAML drug sensitivity and RNA sequencing data were studied to reveal transcriptomic markers of venetoclax resistance and sensitivity. RNA sequencing data of diagnostic, relapsed, refractory AML samples (N = 124) was used to build molecular signatures of drug responses and calculate cell populations scores using the CybersortX algorithm. Four venetoclax-resistant AML cell lines obtained by gradual exposure to 10-1000 nM of venetoclax were used for testing drug combinations by flow cytometry and Annexin V/7-AAD staining. Western blot analysis was used to measure protein levels of the BCL-family and DNA-damage repair pathway. Results: We demonstrate that sensitivity to venetoclax and navitoclax correlates with sensitivity to the HDAC inhibitors belinostat, panobinostat and vorinostat in samples from AML patients with relapsed disease (N = 53). However, no significant correlation between these drugs was observed in the diagnostic samples (N = 51). We show that venetoclax resistant samples in the BeatAML dataset have higher HDAC4 expression, but lower HDAC6 and HDAC2 levels. Using our RNA sequencing data, we depict correlation between the expression of HDAC2, HDAC4, and HDAC6 and the sensitivity to venetoclax and navitoclax. We show that venetoclax-resistant cell lines (OCI-AML3, NOMO-1 and NB-4) do not depend on BCL2, but are very sensitive to BCL2L1, MCL1 and HDAC3 knockout. We demonstrate that BCL2/BCL-xL and HDAC inhibitors target different cell populations: high LSPC-Primed score defines the sensitivity to venetoclax and navitoclax, whilst elevated ProMono-like score – to HDAC inhibitors. Treatment of venetoclax-resistant cell lines (MOLM-13 and HL-60) with non-toxic concentrations of HDAC inhibitors in the presence of 100 nM venetoclax or navitoclax results in the massive death of cells. Finally, we show that dual action of HDAC and BCL-2 inhibitors is realized through DNA-damage activity. Summary/Conclusion: We demonstrated that small doses of HDAC inhibitors, non-toxic as a single agent, can resensitize AML cells to venetoclax and navitoclax by induction of irreversible DNA damage. Keywords: Venetoclax, Acute myeloid leukemia, Histone deacetylase inhibitor